Transcript Fats and Fatty Acid Analysis

A Primer on Fatty Acids and Analyses
Mike Dugan
Meat Lipid Scientist
AAFC-Lacombe
• Fat in beef is needed for
–Flavour
–Tenderness
–Nutritional value
Meat Lipid studies try to understand and manipulate
the feed to meat lipid conversion to
improve fatty acid profiles
• Fats in beef are composed of phospholipids
and triglycerides.
• Phospholipids are found in membranes and
serve structural roles.
• Triglycerides are found in marbling fat and
basically store energy.
Cell Membrane
• Phospholipids have a
glycerol backbone, a polar
phosphate group and 2
fatty acids.
Fatty Acid
Fatty Acid
P
Glycerol
• Triglycerides have a glycerol
backbone and 3 fatty acids
Glycerol
Fatty Acid
ex. Choline
Fatty Acid
Fatty Acid
• Fatty acids are mostly made up of carbon and
hydrogen
Hydrogen
Carbon
Hydrogen can bond to
Carbon
Carbon
can bond to Carbon
As a single or double bond
• Fatty acids are made up of a carbon chain
with an acid group (carboxyl) attached at one
end.
Acid
• when the rest of the bonds are taken up by
hydrogen it a saturated fatty acid (SFA)
• With one double bond it’s a monounsaturated
fatty acid (MUFA)
• With more than one double bond it’s a
polyunsaturated fatty acid (PUFA)
cis
• Double bonds can
have 2 configs.
• hydrogen same
side it’s cis
• hydrogen on
opposite sides
trans.
trans
• A cis double bond
bends the molecule,
• Fatty acids can’t
pack together
closely, decreases
melting and boiling
point
• Trans double bonds
put a kink in the
fatty acid
• FAs can pack
together, has
properties similar
to saturated fatty
acids.
cis
trans
• Fatty acids are usually referred to by their
trivial name or by chemical short-hand
• Common trivial names:
Saturated
MUFA
Stearic
Oleic
Palmitic
Vaccenic
PUFA
CLA
Linoleic (omega-6) Rumenic
Linolenic, EPA, DHA
(all omega-3)
• There are 2 common shorthand systems
• The Delta System – numbers carbons from the
acid (or delta end)
• The Omega System – numbers carbons from
the methyl (or omega end).
Linoleic acid
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
delta
Delta System
omega
c9,c12-18:2
c = cis
Linolenic acid
1
2
3
4
5
6
delta
Delta System
7
8
9 10 11 12 13 14 15 16 17 18
omega
c9,c12,c15-18:3
Vaccenic Acid
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
delta
omega
Delta System
t11-18:1
t = trans
Rumenic Acid
(main natural type or isomer of CLA)
1
2
3
4
5
6
7
8
delta
Delta System
9 10 11 12 13 14 15 16 17 18
omega
c9,t11-18:2
2 double bonds
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
2 carbons
• CLA refers to a group of fatty acids
• CLA’s have 18 carbons & 2 double bonds
separated by 2 carbons
• Double bonds can be found at different places
along the carbons chain
• Besides Rumenic Acid (9c,11t-18:2), CLAs that
can be found in beef include:
t7,c9-18:2
t8,c10-18:2
t10,c12-18:2
t11,c13-18:2
c12,t14-18:2
t7,t9-18:2
t8,t10-18:2
t9,t11-18:2
t10,t12-18:2
t11,t13-18:2
t12,t14-18:2
c7,c9-18:2
c8,c10-18:2
c9,c11-18:2
c10,c12-18:2
c11,c13-18:2
c12,c14-18:2
• Second shorthand system: The Omega System
• Fatty acids named using this system:
– have all double bonds in cis configuration
– adjacent double bonds are separated by 3 carbons
(methylene interrupted)
– The system works for most fatty acids synthesized
by plants and animals
– The system makes it easy to identify related series
of fatty acids (omega-6 and omega-3 fatty acids)
Linoleic acid
Delta System
c9,c12-18:2
3 15
4 14
5 13
6 12
7 11
8 10
9 10
1 17
2 16
9 11
7 13
6 14
5 15
4 16
2 18
18
8 12
3 17
1
delta
omega
Omega System
18:2n-6
This stands for:
18 carbons: 2 double bonds – first double bond at omega-6 carbon
Linolenic acid
Delta System c9,c12,c15-18:3
3 15
4 14
5 13
6 12
7 11
8 10
9 10
9 11
7 13
6 14
5 15
4 16
2 18
1 17
2 16
18
8 12
3 17
1
delta
omega
Omega System
18:3n-3
• Plants and animals can add double bonds.
Plants
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18
delta
omega
Animals
Required
• The two essential fatty acids are:
Linoleic acid
18:2n-6
Used to make LC
Omega-6 fatty acids
Arachidonic acid
20:4n-6
Linolenic acid
18:3n-3
Used to make LC
Omega-3 fatty acids
EPA 20:5n-3
DPA 22:5n-3
DHA 22:6n-3
Beef Lipid Analyses
-1-2-3First cut, grind and Then homogenize Filter & add water
mix to get a with organic solvent
to separate out
representative (2:1 CHCl :CH OH)
the pure lipids
3
3
sample
To analyze individual fatty acids…
Methanol
Lipids
+
Acid or Base
Fatty Acid Methyl Ester (FAME)
GC and HPLC analysis
• Base catalyst works well for backfat because it
contains mostly triglyceride.
• Base catalyst doesn’t work for all meat lipid
classes (FFA, SM, DMA).
• Acid catalysts are a problem in meat when
analyzing conjugated linoleic acid (CLA).
• For meat we combine results from analyses of
separate acid and base methylations.
For GC Analysis
Inject on to column
Increase oven temperature
• On the column: separate based on boiling
point and polarity
– Short chains move faster than long chains
– Saturated move faster than unsaturated
– Trans move faster than cis
Flame
Ionization
Detector
•
If you’d like to analyse the MAJOR fatty acids
in beef, this can be done with
–
–
•
Acid catalyzed methylation
One 15 minute GC analysis with automated peak
measurements
To COMPREHENSIVELY analyze beef fatty acids
–
–
–
–
Acid and base methylation
3 separate GC analyses
1 HPLC analyses
6 hours of machine time and time to make sure
the smaller peaks are identified and measured
correctly
• In a feed sample we typically measure about 15
fatty acids
Saturates
12:0
14:0
16:0
18:0
20:0
22:0
24:0
MUFA
c9-16:1
c9-18:1
c11-18:1
c11-20:1
c13-22:1
PUFA
18:2n-6
18:3n-3
20:2n-6
• In a beef sample we analyse ~80 fatty acids
Saturates
Odd chain
Branched chain
Trans-MUFA
Cis-MUFA
C10:0
C12:0
C14:0
C16:0
C18:0
C20:0
C22:0
C24:0
C13:0
C15:0
C17:0
C19:0
C14:0iso
C15:0iso
C15:0ai
C16:0iso
C17:0iso
C17:0ai
C18:0iso
MUFA
PUFA
c9-14:1
C18:2n-6
c9-16:1
C18:3n-6
c9-18:1
C18:3n-3
c9-20:1
C20:2n-6
c11-20:1
C20:3n-6
c13-22:1
C20:4n-6
t6-t7-16:1 C20:5n-3
t10-16:1
C22:3n-3
t11/t12-16:1 C22:4n-6
t6-t8-18:1 C22:5n-3
t9-18:1
C22:6n-3
t10-18:1
t9c11-CLA
t11-18:1
c9,t11-CLA
t12-18:1
t8,c10-CLA
t13-t14-18:1 t7,c9-CLA
t15-18:1
t12,t14-CLA
t16-18:1
t11,t13-CLA
c9-15:1
t10,t12-CLA
c10-16.1
t9,t11-CLA
c11-16:1
t8,t10-CLA
c12-16:1
t7,t9-CLA
c13-16:1
t11,c13-CLA
c5-17:1
c11,t13-CLA
c7-17:1
t10,c12-CLA
c9-17:1
c9,c11-CLA
c6-c8-18:1 t9t12-18:2
c11-18:1
c9t13-/t8c12-18:2
c12-18:1
t8c13-18:2
c13-18:1
t11c15-18:2
c14-18:1
c9c15-18:2
c15-18:1
Feed or Animal
Bacterial fatty acids
Intermediates of
PUFA hydrogenation
CLA isomers
Other dienes come
from linolenic acid
Why do bacteria hydrogenate?
• PUFA are toxic to bacteria
• So bacteria rapidly hydrogenate linoleic
(18:2n-6) and linolenic acid (18:3n-3) to 18:0
• This goes to completion unless PUFA
somehow protected or hydrogenation
inhibited.
• In most common feeds, >85-95% of the PUFA
are completely hydrogenated.
• This presents a challenge or perhaps a
tremendous opportunity…
• So when measuring fatty acids:
– You buy a standard that has the same fatty acids
your sample has.
– You run your sample and standard on GC.
– You use your standard to identify and measure
the fatty acids in your sample.
– This works well for common fatty acids and when
fatty acids separate well on chromatograms.
• Problems arise with measuring beef fatty acids
because:
– trans-18:1 isomers are difficult to separate
and can overlap with cis-18:1 isomers.
– Many CLA isomers cannot be separated using
GC and you have to use HPLC.
– Standards for most of the hydro. products
are not commercially available.
– You have to use literature reports,
experience, and complementary analyses to
piece together which peaks are which.
• Early studies using comprehensive trans and
CLA analysis of beef indicated most trans-18:1
was vaccenic acid (t11-18:1) and most CLA
was rumenic acid (c9,t11-18:2)
• This created some problems:
– Diets fed during these studies were forage based.
– People assumed results would be similar when
any diets were fed.
– In many instances, people used and still use
methods that don’t separate individual isomers
and assume all trans is vaccenic and all CLA is
rumenic acid.
• Cattle get essential fatty acids from the diet
• In general:
– forages are a source of linolenic acid (omega-3)
– grains are a souce of linoleic acid (omega-6)
– oilseeds including sunflower and safflower have
higher levels of linoleic (omega-6)
– Flax has a high level of linolenic acid (omega-3).
– algae, fish oils and fish meals have high levels of
long chain omega-3’s.
P
• When cattle eat,
rumen bacteria
rapidly hydrolyse
lipids to release
free fatty acids.
• In a few steps
bacteria shift
double bonds and
then add hydrogen
Choline
CLA
Hydrogen
Trans-18:1
Hydrogen
Stearic acid (18:0)
• For years intermediates in hydrogenation like
CLA were ignored.
• In the late 1970’s Mike Pariza’s group from
the University of Wisconsin found:
– CLA from beef protected against cancer
– synthetic CLA reduced body fat
• This led to a number of research projects
studying the effects of CLA and how to increase
levels in beef and dairy products.
• From 1995-2003 my research focused on pork
• AAFC already had people working with beef
lipids and I was happy to work with pork.
• We did some of the first work feeding CLA to
pigs to show it reduces body fat and increases
lean.
• I was a post doc with John Kramer in 1995 and
we’ve worked on methods for trans and CLA
isomer analyses over the past 10-15 years.
• From 2002 to 2004, pork
research was interrupted at
Lacombe due to barn
renovations.
• In 2003 I had the opportunity
to analyse some muskox and
compared these to
conventionally finished beef.
• From the literature we expected both cattle and
muskox would have mostly rumenic and
vaccenic acid as hydrogenation products:
PUFA
Rumenic acid (c9,t11-18:2)
Vaccenic acid (t11-18:1)
• But found all trans isn’t vaccenic and all CLA
isn’t rumenic acid.
• For our current analyses, we use the techniques
developed when working with pork, dairy and
muskox/beef samples.
• First we do one GC analysis with a 175C plateau
which gets most of the fatty acids.
But trans 18:1’s don’t separate well
unkn-diene
c9,c15-18:2
t11c15-18:2
t8c12-18:2
c9t12-18:2
c16-18:1
c9t13-18:2
t8c13-18:2
C19:0
c14-18:1
t16-18:1
c15-18:1
c13-18:1
t10-18:1
t11-18:1
C18:2n-6
c11-/t15-18:1
c9-18:1
trans-18:1
c12-18:1
t6-t8-18:1
t9-18:1
• We then do a 150C run to further separate trans18:1’s and 18:3 hydrogentation products.
18:3 hydro products
GC
unkn after t
oleic acid
7t9c
9c,11t
HPLC
t8,c10
unkn-t12,c14
unkn-c12,t14
unkn-after c12,t14
t11,c13
c11,t13
t10,c12
reagent blank
t12,t14
t11,t13
t10,t12
t9,t11
t8,t10
t7,t9
t6,t8
• We do silver-ion HPLC to separate the CLA
isomers not separating by GC
• In the muskox/beef study:
– Beef diet – barley/barley silage with linoleic acid
(18:2n-6) as the most concentrated PUFA.
– Muskox diet – sedges from the arctic tundra with
equal amounts of linoleic and linolenic acid
(18:3n-3).
Figure 2: Conjugated Linoleic Acid (CLA) Composition
of Backfat
0.6
*
Most concentrated in
Beef and Muskox
% of total fatty acids
0.5
Muskox
Beef
0.4
0.3
Beef
Muskox
0.2
*
0.1
*
*
*
*
0
9c11t-CLA
n = 16; * P< 0.05
9t11c-CLA
11t13c-CLA
7t9c-CLA
CLA-Isomer
10t12c-CLA
Total tt-CLA
• Presently no one knows what effects t7,c918:2 or t11,c13-18:2 are in humans BUT levels
are important to know for future ref.
Trans Fatty Acids
• Vaccenic acid (t11-18:1) was the most concentrated
trans fatty acid in muskox but…
• In beef, we found t10-18:1 was the most
concentrated, and it was quite variable
10t
9c
10t
9t
11t
6-8t
12t
13t/14t / 6-8c
11c
11t
6-8t 9t
65
min 66
13c
12c
12t / 6-8c
9c
14c
16t
15c
10c
15t
36
37
38
39
min
• A high level of vaccenic acid (t11-18:1) is good
as animals use this to make rumenic acid
(c9,t11-18:2) but….
• Increased levels of t10-18:1 are not positive
– t10-18:1 has properties similar to industrially
produced trans fats which negatively effect blood
cholesterol levels in animal models.
• First we wanted to see what the extent
of the problem was:
–We took samples from a study
comparing A (youthful) vs D (cow)
grades (from commercial packing
plant).
–We also conducted a retail survey and
analysed striploin, backfat, hamburger
from Calgary and Guelph&Ohio.
• Second we wanted to figure out how to
limit t10-18:1 and reverse this to t11 and
c9,t11-18:2 if possible.
• Our current understanding:
– Grain diets rich in starch are rapidly
fermented in the rumen.
– Rapid fermentation leads to reduce rumen
pH.
– Lack of fibre, high starch and low rumen pH
shift the rumen bacterial population from
t11-18:1 to t10-18:1 producing species.
Low pH
High Grain
Low Roughage
• The D vs A-Grade Study – confirmed results of Muskox
study
Trans Fatty Acids
3.0
% of Total Fatty Acids ACIDS
2.5
b
t6- to t8-18:1
t9-18:1
t10-18:1
t11-18:1
t12-18:1
b
b
b
b
2.0
c
1.5
a
1.0
b
ab
0.5
a
a
a
ab a
a
a
bc a
a
ab
ab
a
a
a
abc
a
ab
b c
0.0
D1
D2
D3
D4
Maturity/Grade
Y1OTM
Y1UTM
b
• Cows (D grades) likely had more forage
than than concentrate in the diet,
yielding more vaccenic acid than t1018:1.
• Youthful over 30 months of age, likely
summered on pasture before a short stay
in the feedlot. Still more vaccenic than
t10-18:1.
• Youthful under 30 months, definite
“shift” from vaccenic to t10-18:1
CLA – rumenic acid main isomer for all
1.0
b
% of Total Fatty Acids
0.9
t8,c10-18:2
t7,c9-18:2
c9,t11-18:2
t11,c13-18:2
t10,c12-18:2
b
b
b
0.8
b
0.7
0.6
a
0.5
0.4
0.3
0.2
0.1
0.0
a
a
b
b
a
D1
a
a
b
a
D2
b a
D3
b
a
a
a
b
b
D4
Maturity/Grade
b
a
a
c
b
Y1OTM
a
a
ab
Y1UTM
• To try and reverse the 10t shift:
– We checked to see if some common
antibiotics might shift the balance
back to t11-18:1.
– We tried adding buffer to the diet
(partly funded by BCRC).
– We tried adding distillers’ grains (i.e.
grain without starch but higher oil
content)(partly funded by BCRC)
– We analyzed grass versus 1-2mo
grain finishing to see when the trans
and CLA profiles would be affected.
– More recently we have unreported
results on the effects of adding
vitamin E.
• From the D versus A Grade study
– We were also interested in enriching omega-3’s in
beef.
– We calculated hamburger from 1 in 20 animals
had the potential to be labelled omega-3 enriched
(300 mg/100g serving).
• From the 2008 Beef Fatty Acid Workshop in
Lacombe:
– We prepared a proposal looking at ways to
increase omega-3’s in mature and youthful
beef.
– We knew pasture/forage feeding could play a
key role in enriching omega-3s.
• This was based on literature reports on the effects of
forage versus concentrate finishing.
• We wanted to start by feeding flax combined with
forage (Red Clover) to protect linolenic acid in the
rumen.
• Positive results were reported from Kansas
(LaBrune et al., 2008) finishing cattle with flax
in the diet:
– 10% flax was fed in a corn based diet for 85 d
increased linolenic acid (18:3n-3) in longissimus
muscle from 0.2% to 2%.
– Fatty acids reported included:
Saturates
C10:0
C12:0
C14:0
C16:0
C18:0
C20:0
C24:0
C11:0
C13:0
C15:0
C17:0
MUFA
C14:1
C16:1
C18:1
C24:1
C15:1
C17:1
PUFA
C18:2n-6
C18:3n-3
C20:3
C20:4
C20:5
-No hydrogenation products
-No trans
-No CLA
-No other 18:3 hydrogenation
products
-No DHA or DPA
• With this critical information missing we felt
a baseline finishing trial feeding flax in a
barley based diet was needed.
– Fed 0 vs 10% flax in a Barley/Hay diet over 90d
Control (0% flax)
c15-18:1
c9t12-18:2
t8c13-18:2
c9t13-18:2
t11c15-18:2
Mostly not reported by
others
Some negative but mostly
unknown effects
t8c12-18:2
c13-18:1
t16/c14-18:1
c12-18:1
c11-18:1
t12-18:1
t13-t14--18:1
c9-c10-/t15-18:1
t6-t8-18:1
t9-18:1
t10-18:1
t11-18:1
10% flax
cis and trans-18:1
18:2n-6
18:3 hydrogenation
products
control
18:3n-3 CLA
c15-18:1
t16/c14-18:1
c13-18:1
c12-18:1
t11-18:1
t10-18:1
t13-+ t14--18:1
t12-18:1
t6-t8-18:1
t9-18:1
t5-18:1
c11-18:1
c9--18:1
Also a different trans
FA profile was found
c9,t11-18:2
t10,c12-18:2
+
(t7,c9-18:2)
Grain
FORAGE
18:2n-6 Linoleic
oil, monensin
t11-18:1
t10-18:1
c9,t11-18:2
+
(t11,c13-18:2)
t11-18:1
CLAs + Other Dienes
t13-t14-18:1
c15-18:1
Grain + Flax
FORAGE
18:3n-3 Linolenic
Major Points
• If we want to increase or decrease 1-2 fatty
acids in beef, we have to:
– Be able to comprehensively analyse the fatty acids.
– Know what happens to the rest of the fatty acid.
• If you don’t do this and you’ve developed a
product:
– You’ll have troubles if negative health effects are
found later.
– You’ll have repeat all your studies and analyses to
see what’s in your product and how to modify it.